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Introduction. Drought stress is the major factor affecting growth, development and production of walnut trees. In Iran, approximately 33 Mha of land is affected by salinization and drought stress. Finding genetic resources tolerant to drought stress at different growth stages is important for such semi-arid regions. Our aim was to understand better the adaptive mechanisms that enable different genotypes of walnut population to survive under drought stress, and to provide some useful clues for walnut tree breeding toward improved drought tolerance with utilization of existing drought-tolerant genetic resources. Materials and methods. To study the mechanism(s) involved in drought tolerance of some Persian walnut genotypes, drought stress was induced using polyethylene glycol-6000 to produce water potentials of 0 Mpa (control), -0.10 MPa, -0.50 MPa, -0.75 MPa, -1.00 MPa, -1.50 MPa and -2.00 MPa. The amount of proline and soluble sugar accumulation in four walnut genotypes (’Panegine(20)’, ’Lara’, ’serr’ and ’Chandler’) were determined after being exposed to the various water potential levels. Results. The rates of seed germination in all genotypes were significantly reduced by low external water potentials. Plants exposed to water stress had a higher amount of soluble sugars in roots and shoots of tolerant genotypes (’Panegine(20)’ and ’Chandler’) and a lower amount of starch in their tissues. These results imply the important roles of soluble sugars as solutes conferring resistance to drought in these genotypes. The free proline levels were also increased in response to drought stress. They were higher in drought-tolerant genotypes than in sensitive ones (’Lara’ and ’serr’). Proline increased more in shoots than in roots. However, the soluble sugar and starch fluctuations were higher in the roots. Conclusion. Our results support a direct correlation between the degree of drought stress and proline content. As a consequence, proline concentrations could be used as a biochemical marker of drought stress level in walnut plants.